Lettuce cultivar Cayucos

ABSTRACT

A lettuce cultivar, designated Cayucos, is disclosed. The invention relates to the seeds of lettuce cultivar Cayucos, to the plants of lettuce cultivar Cayucos and to methods for producing a lettuce plant by crossing the cultivar Cayucos with itself or another lettuce cultivar. The invention further relates to methods for producing a lettuce plant containing in its genetic material one or more transgenes and to the transgenic lettuce plants and plant parts produced by those methods. This invention also relates to lettuce cultivars or breeding cultivars and plant parts derived from lettuce cultivar Cayucos, to methods for producing other lettuce cultivars, lines or plant parts derived from lettuce cultivar Cayucos and to the lettuce plants, varieties, and their parts derived from the use of those methods. The invention further relates to hybrid lettuce seeds, plants, and plant parts produced by crossing cultivar Cayucos with another lettuce cultivar.

BACKGROUND OF THE INVENTION

The present invention relates to a cos lettuce (Lactuca sativa L.)variety designated Cayucos. All publications cited in this applicationare herein incorporated by reference.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possess the traits to meetthe program goals. The goal is to combine in a single variety or hybridan improved combination of desirable traits from the parental germplasm.These important traits may include increased head size and weight,higher seed yield, improved color, resistance to diseases and insects,tolerance to drought and heat, and better agronomic quality.

Practically speaking, all cultivated forms of lettuce belong to thehighly polymorphic species Lactuca sativa that is grown for its ediblehead and leaves. As a crop, lettuce is grown commercially whereverenvironmental conditions permit the production of an economically viableyield. Lettuce is the World's most popular salad. In the United States,the principal growing regions are California and Arizona which produceapproximately 329,700 acres out of a total annual acreage of more than333,300 acres (USDA (2005)). Fresh lettuce is available in the UnitedStates year-round although the greatest supply is from May throughOctober. For planting purposes, the lettuce season is typically dividedinto three categories (i.e., early, mid, and late), with the coastalareas planting from January to August, and the desert regions plantingfrom August to December. Fresh lettuce is consumed nearly exclusively asfresh, raw product and occasionally as a cooked vegetable.

Lactuca sativa is in the Cichoreae tribe of the Asteraceae (Compositae)family. Lettuce is related to chicory, sunflower, aster, dandelion,artichoke, and chrysanthemum. Sativa is one of about 300 species in thegenus Lactuca. There are seven different morphological types of lettuce.The crisphead group includes the iceberg and batavian types. Iceberglettuce has a large, firm head with a crisp texture and a white orcreamy yellow interior. The batavian lettuce predates the iceberg typeand has a smaller and less firm head. The butterhead group has a small,soft head with an almost oily texture. The romaine, also known as coslettuce, has elongated upright leaves forming a loose, loaf-shaped headand the outer leaves are usually dark green. Leaf lettuce comes in manyvarieties, none of which form a head, and include the green oak leafvariety. Latin lettuce looks like a cross between romaine andbutterhead. Stem lettuce has long, narrow leaves and thick, ediblestems. Oilseed lettuce is a type grown for its large seeds that arepressed to obtain oil. Latin lettuce, stem lettuce, and oilseed lettuceare seldom seen in the United States.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, and recurrent selection.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable cultivar. This approach hasbeen used extensively for breeding disease-resistant cultivars. Variousrecurrent selection techniques are used to improve quantitativelyinherited traits controlled by numerous genes. The use of recurrentselection in self-pollinating crops depends on the ease of pollination,the frequency of successful hybrids from each pollination, and thenumber of hybrid offspring from each successful cross.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard, theoverall value of the advanced breeding lines, and the number ofsuccessful cultivars produced per unit of input (e.g., per year, perdollar expended, etc.).

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for at least three years. The best lines are candidatesfor new commercial cultivars. Those still deficient in a few traits areused as parents to produce new populations for further selection.

These processes, which lead to the final step of marketing anddistribution, usually take from ten to twenty years from the time thefirst cross or selection is made. Therefore, development of newcultivars is a time-consuming process that requires precise forwardplanning, efficient use of resources, and a minimum of changes indirection.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of lettuce plant breeding is to develop new, unique, andsuperior lettuce cultivars. The breeder initially selects and crossestwo or more parental lines, followed by repeated selfing and selection,producing many new genetic combinations. The breeder can theoreticallygenerate billions of different genetic combinations via crossing,selfing, and mutations. The breeder has no direct control at thecellular level. Therefore, two breeders will never develop the sameline, or even very similar lines, having the same lettuce traits.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under different geographical,climatic, and soil conditions, and further selections are then madeduring, and at the end of, the growing season. The cultivars that aredeveloped are unpredictable. This unpredictability is because thebreeder's selection occurs in unique environments, with no control atthe DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.The same breeder cannot produce the same line twice by using the exactsame original parents and the same selection techniques. Thisunpredictability results in the expenditure of large research monies todevelop superior lettuce cultivars.

The development of commercial lettuce cultivars requires the developmentof lettuce varieties, the crossing of these varieties, and theevaluation of the crosses. Pedigree breeding and recurrent selectionbreeding methods are used to develop cultivars from breedingpopulations. Breeding programs combine desirable traits from two or morevarieties or various broad-based sources into breeding pools from whichcultivars are developed by selfing and selection of desired phenotypes.The new cultivars are crossed with other varieties and the hybrids fromthese crosses are evaluated to determine which have commercialpotential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents which possess favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁'s or by intercrossing two F₁'s (sib mating). Selection of the bestindividuals is usually begun in the F₂ population. Then, beginning inthe F₃, the best individuals in the best families are selected.Replicated testing of families, or hybrid combinations involvingindividuals of these families, often follows in the F₄ generation toimprove the effectiveness of selection for traits with low heritability.At an advanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror line that is the recurrent parent. The source of the trait to betransferred is called the donor parent. The resulting plant is expectedto have the attributes of the recurrent parent (e.g., cultivar) and thedesirable trait transferred from the donor parent. After the initialcross, individuals possessing the phenotype of the donor parent areselected and repeatedly crossed (backcrossed) to the recurrent parent.The resulting plant is expected to have the attributes of the recurrentparent (e.g., cultivar) and the desirable trait transferred from thedonor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineswith each generation due to failure of some seeds to germinate or someplants to produce at least one seed. As a result, not all of the F₂plants originally sampled in the population will be represented by aprogeny when generation advance is completed.

In addition to phenotypic observations, the genotype of a plant can alsobe examined. There are many laboratory-based techniques available forthe analysis, comparison and characterization of plant genotype; amongthese are Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length polymorphisms (AFLPs), Simple Sequence Repeats(SSRs—which are also referred to as Microsatellites), and SingleNucleotide Polymorphisms (SNPs).

Isozyme Electrophoresis and RFLPs have been widely used to determinegenetic composition. Shoemaker and Olsen (Molecular Linkage Map ofSoybean (Glycine max), pp. 6.131-6.138 in S. J. O'Brien (ed.) GeneticMaps: Locus Maps of Complex Genomes, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1993)) developed a molecular geneticlinkage map that consisted of 25 linkage groups with about 365 RFLP, 11RAPD, three classical markers, and four isozyme loci. See also,Shoemaker, R. C., RFLP Map of Soybean, pp. 299-309, in Phillips, R. L.and Vasil, I. K. (eds.), DNA-Based Markers in Plants, Kluwer AcademicPress, Dordrecht, the Netherlands (1994).

SSR technology is currently the most efficient and practical markertechnology; more marker loci can be routinely used and more alleles permarker locus can be found using SSRs in comparison to RFLPs. Forexample, Diwan and Cregan described a highly polymorphic microsatellitelocus in soybean with as many as 26 alleles. Diwan, N. and Cregan, P.B., Theor. Appl. Genet., 95:22-225 (1997). SNPs may also be used toidentify the unique genetic composition of the invention and progenyvarieties retaining that unique genetic composition. Various molecularmarker techniques may be used in combination to enhance overallresolution.

Molecular markers, which include markers identified through the use oftechniques such as Isozyme Electrophoresis, RFLPs, RAPDs, AP-PCR, DAF,SCARs, AFLPs, SSRs, and SNPs, may be used in plant breeding. One use ofmolecular markers is Quantitative Trait Loci (QTL) mapping. QTL mappingis the use of markers which are known to be closely linked to allelesthat have measurable effects on a quantitative trait. Selection in thebreeding process is based upon the accumulation of markers linked to thepositive effecting alleles and/or the elimination of the markers linkedto the negative effecting alleles from the plant's genome.

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. The markers can also beused to select toward the genome of the recurrent parent and against themarkers of the donor parent. This procedure attempts to minimize theamount of genome from the donor parent that remains in the selectedplants. It can also be used to reduce the number of crosses back to therecurrent parent needed in a backcrossing program. The use of molecularmarkers in the selection process is often called genetic marker enhancedselection or marker-assisted selection. Molecular markers may also beused to identify and exclude certain sources of germplasm as parentalvarieties or ancestors of a plant by providing a means of trackinggenetic profiles through crosses.

Mutation breeding is another method of introducing new traits intolettuce varieties. Mutations that occur spontaneously or areartificially induced can be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates can be increasedby many different means including temperature, long-term seed storage,tissue culture conditions, radiation (such as X-rays, Gamma rays,neutrons, Beta radiation, or ultraviolet radiation), chemical mutagens(such as base analogs like 5-bromo-uracil), antibiotics, alkylatingagents (such as sulfur mustards, nitrogen mustards, epoxides,ethyleneamines, sulfates, sulfonates, sulfones, or lactones), azide,hydroxylamine, nitrous acid, or acridines. Once a desired trait isobserved through mutagenesis the trait may then be incorporated intoexisting germplasm by traditional breeding techniques. Details ofmutation breeding can be found in Principles of Cultivar Development byFehr, Macmillan Publishing Company (1993).

The production of double haploids can also be used for the developmentof homozygous varieties in a breeding program. Double haploids areproduced by the doubling of a set of chromosomes from a heterozygousplant to produce a completely homozygous individual. For example, seeWan, et al., Theor. Appl. Genet., 77:889-892 (1989).

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Principles of Plant Breeding, John Wiley and Son, pp.115-161 (1960); Allard (1960); Simmonds (1979); Sneep, et al. (1979);Fehr (1987); “Carrots and Related Vegetable Umbelliferae,” Rubatzky, V.E., et al. (1999).

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts, as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

Lettuce in general, and leaf lettuce in particular, is an important andvaluable vegetable crop. Thus, a continuing goal of lettuce plantbreeders is to develop stable, high yielding lettuce cultivars that areagronomically sound. To accomplish this goal, the lettuce breeder mustselect and develop lettuce plants with traits that result in superiorcultivars.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described inconjunction with systems, tools, and methods which are meant to beexemplary and illustrative, not limiting in scope. In variousembodiments, one or more of the above-described problems have beenreduced or eliminated, while other embodiments are directed to otherimprovements.

According to the invention, there is provided a novel lettuce cultivardesignated Cayucos. This invention thus relates to the seeds of lettucecultivar Cayucos, to the plants of lettuce cultivar Cayucos, and tomethods for producing a lettuce plant produced by crossing the lettucecultivar Cayucos with itself or another lettuce plant, to methods forproducing a lettuce plant containing in its genetic material one or moretransgenes, and to the transgenic lettuce plants produced by thatmethod. This invention also relates to methods for producing otherlettuce cultivars derived from lettuce cultivar Cayucos and to thelettuce cultivar derived by the use of those methods. This inventionfurther relates to hybrid lettuce seeds and plants produced by crossinglettuce cultivar Cayucos with another lettuce variety.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of lettuce cultivar Cayucos. The tissue culturewill preferably be capable of regenerating plants having essentially allof the physiological and morphological characteristics of the foregoinglettuce plant, and of regenerating plants having substantially the samegenotype as the foregoing lettuce plant. Preferably, the regenerablecells in such tissue cultures will be callus, protoplasts, meristematiccells, cotyledons, hypocotyl, leaves, pollen, embryos, roots, root tips,anthers, pistils, shoots, stems, petiole flowers, and seeds. Stillfurther, the present invention provides lettuce plants regenerated fromthe tissue cultures of the invention.

Another aspect of the invention is to provide methods for producingother lettuce plants derived from lettuce cultivar Cayucos. Lettucecultivars derived by the use of those methods are also part of theinvention.

The invention also relates to methods for producing a lettuce plantcontaining in its genetic material one or more transgenes and to thetransgenic lettuce plant produced by those methods.

In another aspect, the present invention provides for single geneconverted plants of Cayucos. The single transferred gene may preferablybe a dominant or recessive allele. Preferably, the single transferredgene will confer such traits as male sterility, herbicide resistance,insect or pest resistance, modified fatty acid metabolism, modifiedcarbohydrate metabolism, resistance for bacterial, fungal, or viraldisease, male fertility, enhanced nutritional quality, and industrialusage. The single gene may be a naturally occurring lettuce gene or atransgene introduced through genetic engineering techniques.

The invention further provides methods for developing lettuce plants ina lettuce plant breeding program using plant breeding techniquesincluding recurrent selection, backcrossing, pedigree breeding,restriction fragment length polymorphism enhanced selection, geneticmarker enhanced selection, and transformation. Seeds, lettuce plants,and parts thereof, produced by such breeding methods are also part ofthe invention.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference bystudy of the following descriptions.

DEFINITIONS

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. The allele is any of one or more alternative forms of a gene,all of which relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotype of the F₁ hybrid.

Big Vein virus. Big vein is a disease of lettuce caused by LettuceMirafiori Big Vein Virus which is transmitted by the fungus Olpidiumvirulentus, with vein clearing and leaf shrinkage resulting in plants ofpoor quality and reduced marketable value.

Bolting. The premature development of a flowering stalk, and subsequentseed, before a plant produces a food crop. Bolting is typically causedby late planting when temperatures are low enough to cause vernalizationof the plants.

Bremia lactucae. An Oomycete that causes downy mildew in lettuce incooler growing regions.

Core length. Length of the internal lettuce stem measured from the baseof the cut and trimmed head to the tip of the stem.

Corky root. A disease caused by the bacterium Sphingomonassuberifaciens, which causes the entire taproot to become brown, severelycracked, and non-functional.

Cotyledon. One of the first leaves of the embryo of a seed plant;typically one or more in monocotyledons, two in dicotyledons, and two ormore in gymnosperms.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics of the recurrent parent, except for the characteristicsderived from the converted gene.

First water date. The date the seed first receives adequate moisture togerminate. This can and often does equal the planting date.

Gene. As used herein, “gene” refers to a segment of nucleic acid. A genecan be introduced into a genome of a species, whether from a differentspecies or from the same species, using transformation or variousbreeding methods.

Head diameter. Diameter of the cut and trimmed head, sliced vertically,and measured at the widest point perpendicular to the stem.

Head height. Height of the cut and trimmed head, sliced vertically, andmeasured from the base of the cut stem to the cap leaf.

Head weight. Weight of saleable lettuce head, cut and trimmed to marketspecifications.

Lettuce Mosaic virus. A disease that can cause a stunted, deformed, ormottled pattern in young lettuce and yellow, twisted, and deformedleaves in older lettuce.

Maturity date. Maturity refers to the stage when the plants are of fullsize or optimum weight, in marketable form or shape to be of commercialor economic value.

Nasonovia ribisnigri. A lettuce aphid that colonizes the innermostleaves of the lettuce plant, contaminating areas that cannot be treatedeasily with insecticides.

Quantitative Trait Loci. Quantitative Trait Loci (QTL) refers to geneticloci that control to some degree, numerically representable traits thatare usually continuously distributed.

Ratio of head height/diameter. Head height divided by the head diameteris an indication of the head shape; <1 is flattened, 1=round, and >1 ispointed.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

RHS. RHS refers to the Royal Horticultural Society of England whichpublishes an official botanical color chart quantitatively identifyingcolors according to a defined numbering system. The chart may bepurchased from Royal Horticulture Society Enterprise Ltd., RHS Garden;Wisley, Woking; Surrey GU236QB, UK.

Single gene converted. Single gene converted or conversion plant refersto plants which are developed by a plant breeding technique calledbackcrossing or via genetic engineering wherein essentially all of thedesired morphological and physiological characteristics of a line arerecovered in addition to the single gene transferred into the line viathe backcrossing technique or via genetic engineering.

Tip burn. Means a browning of the edges or tips of lettuce leaves thatis a physiological response to a lack of calcium.

Tomato Bushy Stunt. A disease which causes stunting of growth, leafmottling, and deformed or absent fruit.

DETAILED DESCRIPTION OF THE INVENTION

Lettuce cultivar Cayucos is a cos lettuce variety suitable forproduction in Arizona and California of the United States. Additionally,lettuce cultivar Cayucos is resistant to B1:1-27/Nr:0/LMV. It is theresult of numerous generations of plant selections chosen for its threedimensional leaf shape and very late bolting under long day conditions.

The cultivar has shown uniformity and stability for the traits, withinthe limits of environmental influence for the traits. It has beenself-pollinated a sufficient number of generations with carefulattention to uniformity of plant type. The line has been increased withcontinued observation for uniformity. No variant traits have beenobserved or are expected in cultivar Cayucos.

Lettuce cultivar Cayucos has the following morphologic and othercharacteristics, described in Table 1.

TABLE 1 VARIETY DESCRIPTION INFORMATION Plant: Type: Cos lettuceMaturity date: 60 days from wet date in summer sowings Seed: Color:White Light dormancy: None Heat dormancy: None Cotyledon (to fourth leafstage): Shape: Broad Shape of fourth leaf: Elongated Length/width indexof 4th leaf 65/36 × 10 = 18 (L/W × 10): Apex: Entire Base: Moderatelydentate Undulation: Flat Green color: Dark green Anthocyanindistribution: Absent Rolling: Absent Cupping: Uncupped Reflexing: NoneMature Leaves: Margin: Incision depth: Absent Indentation: AbsentUndulation of the apical margin: Absent Green color (at harvestmaturity): Dark Hue of green color of outer leaves: Absent Intensity ofcolor of outer leaves: High Anthocyanin distribution: Absent Size: SmallGlossiness: Dull Blistering: Present Thickness: Thick Trichomes: AbsentPlant (at market stage): Spread of frame leaves:  25.0 cm Butt: Shape:Rounded Midrib: Flattened Core: Diameter at base of head:  3.0 cm Coreheight from base of head to apex:  4.5 cm Bolting: First water date:Aug. 01, 2010 Number of days from first water date 68 days to seed stalkemergence: Time of beginning of bolting: 68 days Class: Relatively slowbolting when grown in long day summer productions Height of mature seedstalk: 125.0 cm Spread of bolter plant:  27.0 cm Bolter leaves: Pointedand bit folded Bolter habit: Medium bolting Primary Regions ofAdaptation: Spring area: California, U.S. Summer area: California, U.S.Autumn area: California, U.S. Winter area: Desert Southwest, Arizona andCentral Valley, California, U.S. Disease/Pest Resistance: Lettuce MosaicVirus: Resistant (common LS1 strain) Downy Mildew (Bremia lactucae):Resistant Bl; 1-27 Pests: Nasonovia ribisnigri: Resistant Nr: 0Physiological stresses: Tipburn: Moderately resistant

Additional methods include, but are not limited to, expression vectorsintroduced into plant tissues using a direct gene transfer method, suchas microprojectile-mediated delivery, DNA injection, electroporation,and the like. More preferably, expression vectors are introduced intoplant tissues by using either microprojectile-mediated delivery with abiolistic device or by using Agrobacterium-mediated transformation.Transformed plants obtained with the protoplasm of the invention areintended to be within the scope of this invention.

Further Embodiments of the Invention

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Any DNA sequences,whether from a different species or from the same species, which areintroduced into the genome using transformation or various breedingmethods, are referred to herein collectively as “transgenes.” Over thelast fifteen to twenty years, several methods for producing transgenicplants have been developed, and the present invention, in particularembodiments, also relates to transformed versions of the claimed line.

Nucleic acids or polynucleotides refer to RNA or DNA that is linear orbranched, single or double stranded, or a hybrid thereof. The term alsoencompasses RNA/DNA hybrids. These terms also encompass untranslatedsequence located at both the 3′ and 5′ ends of the coding region of thegene: at least about 1000 nucleotides of sequence upstream from the 5′end of the coding region and at least about 200 nucleotides of sequencedownstream from the 3′ end of the coding region of the gene. Less commonbases, such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine,and others can also be used for antisense, dsRNA, and ribozyme pairing.For example, polynucleotides that contain C-5 propyne analogues ofuridine and cytidine have been shown to bind RNA with high affinity andto be potent antisense inhibitors of gene expression. Othermodifications, such as modification to the phosphodiester backbone, orthe 2′-hydroxy in the ribose sugar group of the RNA can also be made.The antisense polynucleotides and ribozymes can consist entirely ofribonucleotides, or can contain mixed ribonucleotides anddeoxyribonucleotides. The polynucleotides of the invention may beproduced by any means, including genomic preparations, cDNApreparations, in vitro synthesis, RT-PCR, and in vitro or in vivotranscription.

Plant transformation involves the construction of an expression vectorthat will function in plant cells. Such a vector comprises DNAcomprising a gene under control of, or operatively linked to, aregulatory element (for example, a promoter). The expression vector maycontain one or more such operably linked gene/regulatory elementcombinations. The vector(s) may be in the form of a plasmid, and can beused alone or in combination with other plasmids, to provide transformedlettuce plants using transformation methods as described below toincorporate transgenes into the genetic material of the lettuceplant(s).

Expression Vectors for Lettuce Transformation: Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element (for example, a promoter) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or an herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from transposonTn5, which when placed under the control of plant regulatory signalsconfers resistance to kanamycin. Fraley, et al., PNAS, 80:4803 (1983).Another commonly used selectable marker gene is the hygromycinphosphotransferase gene which confers resistance to the antibiotichygromycin. Vanden Elzen, et al., Plant Mol. Biol., 5:299 (1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant. Hayford, et al., Plant Physiol.,86:1216 (1988); Jones, et al., Mol. Gen. Genet., 210:86 (1987); Svab, etal., Plant Mol. Biol., 14:197 (1990); Hille, et al., Plant Mol. Biol.,7:171 (1986). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate, or bromoxynil. Comai, etal., Nature, 317:741-744 (1985); Gordon-Kamm, et al., Plant Cell,2:603-618 (1990); and Stalker, et al., Science, 242:419-423 (1988).

Selectable marker genes for plant transformation that are not ofbacterial origin include, for example, mouse dihydrofolate reductase,plant 5-enolpyruvylshikimate-3-phosphate synthase, and plantacetolactate synthase. Eichholtz, et al., Somatic Cell Mol. Genet.,13:67 (1987); Shah, et al., Science, 233:478 (1986); and Charest, etal., Plant Cell Rep., 8:643 (1990).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include α-glucuronidase (GUS),α-galactosidase, luciferase and chloramphenicol, acetyltransferase.Jefferson, R. A., Plant Mol. Biol., 5:387 (1987); Teeri, et al., EMBOJ., 8:343 (1989); Koncz, et al., PNAS, 84:131 (1987); and DeBlock, etal., EMBO J., 3:1681 (1984).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissues are available. Molecular Probes,Publication 2908, IMAGENE GREEN, pp. 1-4 (1993) and Naleway, et al., J.Cell Biol., 115:151a (1991). However, these in vivo methods forvisualizing GUS activity have not proven useful for recovery oftransformed cells because of low sensitivity, high fluorescentbackgrounds, and limitations associated with the use of luciferase genesas selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie, et al., Science, 263:802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Expression Vectors for Lettuce Transformation: Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element (for example, a promoter).Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred.”Promoters which initiate transcription only in certain tissue arereferred to as “tissue-specific.” A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters:

An inducible promoter is operably linked to a gene for expression inlettuce. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in lettuce. With an inducible promoter, therate of transcription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward,et al., Plant Mol. Biol., 22:361-366 (1993). Exemplary induciblepromoters include, but are not limited to, that from the ACEI systemwhich responds to copper (Melt, et al., PNAS, 90:4567-4571 (1993)); In2gene from maize which responds to benzenesulfonamide herbicide safeners(Hershey, et al., Mol. Gen. Genet., 227:229-237 (1991) and Gatz, et al.,Mol. Gen. Genet., 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz, etal., Mol. Gen. Genet., 227:229-237 (1991)). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena,et al., PNAS, 88:0421 (1991).

B. Constitutive Promoters:

A constitutive promoter is operably linked to a gene for expression inlettuce or the constitutive promoter is operably linked to a nucleotidesequence encoding a signal sequence which is operably linked to a genefor expression in lettuce.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell, et al., Nature, 313:810-812 (1985)) and the promoters from suchgenes as rice actin (McElroy, et al., Plant Cell, 2:163-171 (1990));ubiquitin (Christensen, et al., Plant Mol. Biol., 12:619-632 (1989) andChristensen, et al., Plant Mol. Biol., 18:675-689 (1992)); pEMU (Last,et al., Theor. Appl. Genet., 81:581-588 (1991)); MAS (Velten, et al.,EMBO J., 3:2723-2730 (1984)) and maize H3 histone (Lepetit, et al., Mol.Gen. Genet., 231:276-285 (1992) and Atanassova, et al., Plant J., 2(3):291-300 (1992)). The ALS promoter, Xbal/Ncol fragment 5′ to theBrassica napus ALS3 structural gene (or a nucleotide sequence similarityto said Xbal/Ncol fragment), represents a particularly usefulconstitutive promoter. See PCT Application No. WO 96/30530.

C. Tissue-Specific or Tissue-Preferred Promoters:

A tissue-specific promoter is operably linked to a gene for expressionin lettuce. Optionally, the tissue-specific promoter is operably linkedto a nucleotide sequence encoding a signal sequence which is operablylinked to a gene for expression in lettuce. Plants transformed with agene of interest operably linked to a tissue-specific promoter producethe protein product of the transgene exclusively, or preferentially, ina specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai, et al., Science, 23:476-482(1983) and Sengupta-Gopalan, et al., PNAS, 82:3320-3324 (1985)); aleaf-specific and light-induced promoter such as that from cab orrubisco (Simpson, et al., EMBO J., 4(11):2723-2729 (1985) and Timko, etal., Nature, 318:579-582 (1985)); an anther-specific promoter such asthat from LAT52 (Twell, et al., Mol. Gen. Genet., 217:240-245 (1989)); apollen-specific promoter such as that from Zm13 (Guerrero, et al., Mol.Gen. Genet., 244:161-168 (1993)) or a microspore-preferred promoter suchas that from apg (Twell, et al., Sex. Plant Reprod., 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall, ormitochondrion, or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample, Becker, et al., Plant Mol. Biol., 20:49 (1992); Close, P. S.,Master's Thesis, Iowa State University (1993); Knox, C., et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley,” Plant Mol. Biol., 9:3-17 (1987); Lerner, et al., PlantPhysiol., 91:124-129 (1989); Fontes, et al., Plant Cell, 3:483-496(1991); Matsuoka, et al., PNAS, 88:834 (1991); Gould, et al., J. Cell.Biol., 108:1657 (1989); Creissen, et al., Plant J., 2:129 (1991);Kalderon, et al., A short amino acid sequence able to specify nuclearlocation, Cell, 39:499-509 (1984); and Steifel, et al., Expression of amaize cell wall hydroxyproline-rich glycoprotein gene in early leaf androot vascular differentiation, Plant Cell, 2:785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem., 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is lettuce. In anotherpreferred embodiment, the biomass of interest is seed. For therelatively small number of transgenic plants that show higher levels ofexpression, a genetic map can be generated, primarily via conventionalRFLP, PCR, and SSR analysis, which identifies the approximatechromosomal location of the integrated DNA molecule. For exemplarymethodologies in this regard, see Methods in Plant Molecular Biology andBiotechnology, Glick and Thompson Eds., 269:284, CRC Press, Boca Raton(1993). Map information concerning chromosomal location is useful forproprietary protection of a subject transgenic plant. If unauthorizedpropagation is undertaken and crosses made with other germplasm, the mapof the integration region can be compared to similar maps for suspectplants, to determine if the latter have a common parentage with thesubject plant. Map comparisons would involve hybridizations, RFLP, PCR,SSR, and sequencing, all of which are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

A. Genes that Confer Resistance to Pests or Disease and that Encode:

1. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant line can be transformed with a clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example, Jones, et al., Science, 266:789(1994) (cloning of the tomato Cf-9 gene for resistance to Cladosporiumfulvum); Martin, et al., Science, 262:1432 (1993) (tomato Pto gene forresistance to Pseudomonas syringae pv. tomato encodes a protein kinase);and Mindrinos, et al., Cell, 78:1089 (1994) (Arabidopsis RSP2 gene forresistance to Pseudomonas syringae).

2. A Bacillus thuringiensis protein, a derivative thereof, or asynthetic polypeptide modeled thereon. See, for example, Geiser, et al.,Gene, 48:109 (1986), who disclose the cloning and nucleotide sequence ofa Bt δ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxingenes can be purchased from American Type Culture Collection, Manassas,Va., for example, under ATCC Accession Nos. 40098, 67136, 31995, and31998.

3. A lectin. See, for example, the disclosure by Van Damme, et al.,Plant Mol. Biol., 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

4. A vitamin-binding protein such as avidin. See PCT Application No. US93/06487, the contents of which are hereby incorporated by reference.The application teaches the use of avidin and avidin homologues aslarvicides against insect pests.

5. An enzyme inhibitor, for example, a protease or proteinase inhibitor,or an amylase inhibitor. See, for example, Abe, et al., J. Biol. Chem.,262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor); Huub, et al., Plant Mol. Biol., 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I); and Sumitani,et al., Biosci. Biotech. Biochem., 57:1243 (1993) (nucleotide sequenceof Streptomyces nitrosporeus α-amylase inhibitor).

6. An insect-specific hormone or pheromone, such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock, et al., Nature, 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

7. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem., 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor) and Pratt, etal., Biochem. Biophys. Res. Comm., 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also, U.S. Pat. No. 5,266,317 toTomalski, et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

8. An insect-specific venom produced in nature, by a snake, a wasp, etc.For example, see Pang, et al., Gene, 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

9. An enzyme responsible for a hyper-accumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivative,or another non-protein molecule with insecticidal activity.

10. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase, and a glucanase, whether natural or synthetic. See PCTApplication No. WO 93/02197 in the name of Scott, et al., whichdiscloses the nucleotide sequence of a callase gene. DNA molecules whichcontain chitinase-encoding sequences can be obtained, for example, fromthe ATCC under Accession Nos. 39637 and 67152. See also, Kramer, et al.,Insect Biochem. Mol. Biol., 23:691 (1993), who teach the nucleotidesequence of a cDNA encoding tobacco hornworm chitinase, and Kawalleck,et al., Plant Mol. Biol., 21:673 (1993), who provide the nucleotidesequence of the parsley ubi4-2 polyubiquitin gene.

11. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella, et al., Plant Mol. Biol., 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess,et al., Plant Physiol., 104:1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

12. A hydrophobic moment peptide. See PCT Application No. WO 95/16776(disclosure of peptide derivatives of tachyplesin which inhibit fungalplant pathogens) and PCT Application No. WO 95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

13. A membrane permease, a channel former, or a channel blocker. Forexample, see the disclosure of Jaynes, et al., Plant Sci., 89:43 (1993),of heterologous expression of a cecropin-β, lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

14. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy, et al., Ann. Rev. Phytopathol.,28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus, and tobacco mosaic virus. Id.

15. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. SeeTaylor, et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions, Edinburgh, Scotland (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

16. A virus-specific antibody. See, for example, Tavladoraki, et al.,Nature, 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

17. A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo-α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient released by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See Lamb, et al., Bio/technology,10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubart,et al., Plant J., 2:367 (1992).

18. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann, et al., Bio/technology, 10:305 (1992), have shownthat transgenic plants expressing the barley ribosome-inactivating genehave an increased resistance to fungal disease.

19. A lettuce mosaic potyvirus (LMV) coat protein gene introduced intoLactuca sativa in order to increase its resistance to LMV infection. SeeDinant, et al., Mol. Breeding, 3:1, 75-86 (1997).

Any of the above listed disease or pest resistance genes (1-19) can beintroduced into the claimed lettuce cultivar through a variety of meansincluding but not limited to transformation and crossing.

B. Genes that Confer Resistance to an Herbicide:

1. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee, etal., EMBO J., 7:1241 (1988) and Miki, et al., Theor. Appl. Genet.,80:449 (1990), respectively.

2. Glyphosate (resistance conferred by mutant5-enolpyruvlshikimate-3-phosphate synthase (EPSPS) and aroA genes,respectively) and other phosphono compounds, such as glufosinate(phosphinothricin acetyl transferase (PAT), dicamba and Streptomyceshygroscopicus phosphinothricin-acetyl transferase PAT bar genes), andpyridinoxy or phenoxy proprionic acids and cyclohexones (ACCaseinhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 toShah, et al., which discloses the nucleotide sequence of a form of EPSPSwhich can confer glyphosate resistance. A DNA molecule encoding a mutantaroA gene can be obtained under ATCC Accession No. 39256, and thenucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.4,769,061 to Comai. See also, Umaballava-Mobapathie in TransgenicResearch, 8:1, 33-44 (1999) that discloses Lactuca sativa resistant toglufosinate. European Patent Application No. 0 333 033 to Kumada, etal., and U.S. Pat. No. 4,975,374 to Goodman, et al., disclose nucleotidesequences of glutamine synthetase genes which confer resistance toherbicides, such as L-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in EuropeanApplication No. 0 242 246 to Leemans, et al. DeGreef, et al.,Bio/technology, 7:61 (1989), describe the production of transgenicplants that express chimeric bar genes coding for phosphinothricinacetyl transferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cyclohexones, such as sethoxydim andhaloxyfop, are the Acc1-S1, Acc1-S2, and Acc1-S3 genes described byMarshall, et al., Theor. Appl. Genet., 83:435 (1992).

3. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibilla, et al.,Plant Cell, 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes, et al., Biochem. J.,285:173 (1992).

4. Acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants. See Hattori, et al., Mol. Gen.Genet., 246:419 (1995). Other genes that confer tolerance to herbicidesinclude a gene encoding a chimeric protein of rat cytochrome P4507A1 andyeast NADPH-cytochrome P450 oxidoreductase (Shiota, et al., PlantPhysiol., 106:17 (1994)), genes for glutathione reductase and superoxidedismutase (Aono, et al., Plant Cell Physiol., 36:1687 (1995)), and genesfor various phosphotransferases (Datta, et al., Plant Mol. Biol., 20:619(1992)).

5. Protoporphyrinogen oxidase (protox) is necessary for the productionof chlorophyll, which is necessary for all plant survival. The protoxenzyme serves as the target for a variety of herbicidal compounds. Theseherbicides also inhibit growth of all the different species of plantspresent, causing their total destruction. The development of plantscontaining altered protox activity which are resistant to theseherbicides are described in U.S. Pat. Nos. 6,288,306, 6,282,837,5,767,373, and International Publication WO 01/12825.

Any of the above listed herbicide genes (1-5) can be introduced into theclaimed lettuce cultivar through a variety of means including, but notlimited to, transformation and crossing.

C. Genes that Confer or Contribute to a Value-Added Trait, such as:

1. Increased iron content of the lettuce, for example, by introducinginto a plant a soybean ferritin gene as described in Goto, et al., ActaHorticulturae., 521, 101-109 (2000).

2. Decreased nitrate content of leaves, for example, by introducing intoa lettuce a gene coding for a nitrate reductase. See, for example,Curtis, et al., Plant Cell Rep., 18:11, 889-896 (1999).

3. Increased sweetness of the lettuce by introducing a gene coding formonellin that elicits a flavor 100,000 times sweeter than sugar on amolar basis. See Penarrubia, et al., Bio/technology, 10:561-564 (1992).

4. Modified fatty acid metabolism, for example, by introducing into aplant an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon, et al., PNAS, 89:2625 (1992).

5. Modified carbohydrate composition effected, for example, byintroducing into plants a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza, et al., J. Bacteriol., 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene); Steinmetz, et al., Mol. Gen. Genet., 20:220(1985) (nucleotide sequence of Bacillus subtilis levansucrase gene);Pen, et al., Bio/technology, 10:292 (1992) (production of transgenicplants that express Bacillus lichenifonnis α-amylase); Elliot, et al.,Plant Mol. Biol., 21:515 (1993) (nucleotide sequences of tomatoinvertase genes); Søgaard, et al., J. Biol. Chem., 268:22480 (1993)(site-directed mutagenesis of barley α-amylase gene); and Fisher, etal., Plant Physiol., 102:1045 (1993) (maize endosperm starch branchingenzyme II).

D. Genes that Control Male-Sterility:

1. Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN-Ac-PPT. See International Publication WO 01/29237.

2. Introduction of various stamen-specific promoters. See InternationalPublications WO 92/13956 and WO 92/13957.

3. Introduction of the barnase and the barstar genes. See Paul, et al.,Plant Mol. Biol., 19:611-622 (1992).

Methods for Lettuce Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Mild, et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glickand Thompson Eds., CRC Press, Inc., Boca Raton, pp. 67-88 (1993). Inaddition, expression vectors and in vitro culture methods for plant cellor tissue transformation and regeneration of plants are available. See,for example, Gruber, et al., “Vectors for Plant Transformation” inMethods in Plant Molecular Biology and Biotechnology, Glick and ThompsonEds., CRC Press, Inc., Boca Raton, pp. 89-119 (1993).

A. Agrobacterium-Mediated Transformation:

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch, et al., Science, 227:1229 (1985); Curtis, et al., Journal ofExperimental Botany, 45:279, 1441-1449 (1994); Torres, et al., PlantCell Tissue and Organ Culture, 34:3, 279-285 (1993); and Dinant, et al.,Molecular Breeding, 3:1, 75-86 (1997). A. tumefaciens and A. rhizogenesare plant pathogenic soil bacteria which genetically transform plantcells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes,respectively, carry genes responsible for genetic transformation of theplant. See, for example, Kado, C. I., Crit. Rev. Plant Sci., 10:1(1991). Descriptions of Agrobacterium vector systems and methods forAgrobacterium-mediated gene transfer are provided by Gruber, et al.,supra, Miki, et al., supra, and Moloney, et al., Plant Cell Rep., 8:238(1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7, 1997.

B. Direct Gene Transfer:

Several methods of plant transformation collectively referred to asdirect gene transfer have been developed as an alternative toAgrobacterium-mediated transformation. A generally applicable method ofplant transformation is microprojectile-mediated transformation whereinDNA is carried on the surface of microprojectiles measuring 1 μm to 4μm. The expression vector is introduced into plant tissues with abiolistic device that accelerates the microprojectiles to speeds of 300m/s to 600 m/s which is sufficient to penetrate plant cell walls andmembranes. Russell, D. R., et al., Plant Cell Rep., 12 (3, January),165-169 (1993); Aragao, F. J. L., et al., Plant Mol. Biol., 20 (2,October), 357-359 (1992); Aragao, F. J. L., et al., Plant Cell Rep., 12(9, July), 483-490 (1993); Aragao, Theor. Appl. Genet., 93:142-150(1996); Kim, J., Minamikawa, T., Plant Sci., 117:131-138 (1996);Sanford, et al., Part. Sci. Technol., 5:27 (1987); Sanford, J. C.,Trends Biotech., 6:299 (1988); Klein, et al., Bio/technology, 6:559-563(1988); Sanford, J. C., Physiol. Plant, 7:206 (1990); Klein, et al.,Bio/technology, 10:268 (1992).

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang, et al., Bio/technology, 9:996 (1991).Alternatively, liposome and spheroplast fusion have been used tointroduce expression vectors into plants. Deshayes, et al., EMBO J.,4:2731 (1985) and Christou, et al., PNAS, 84:3962 (1987). Direct uptakeof DNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol, orpoly-L-ornithine has also been reported. Hain, et al., Mol. Gen. Genet.,199:161 (1985) and Draper, et al., Plant Cell Physiol., 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Saker, M., Kuhne, T., Biologia Plantarum, 40(4):507-514(1997/98); Donn, et al., In Abstracts of VIIth International Congress onPlant Cell and Tissue Culture IAPTC, A2-38, p. 53 (1990); D'Halluin, etal., Plant Cell, 4:1495-1505 (1992); and Spencer, et al., Plant Mol.Biol., 24:51-61 (1994). See also Chupean, et al., Bio/technology, 7:5,503-508 (1989).

Following transformation of lettuce target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic line. The transgenic line could then be crossedwith another (non-transformed or transformed) line in order to produce anew transgenic lettuce line. Alternatively, a genetic trait which hasbeen engineered into a particular lettuce cultivar using the foregoingtransformation techniques could be introduced into another line usingtraditional backcrossing techniques that are well known in the plantbreeding arts. For example, a backcrossing approach could be used tomove an engineered trait from a public, non-elite inbred line into anelite inbred line, or from an inbred line containing a foreign gene inits genome into an inbred line or lines which do not contain that gene.As used herein, “crossing” can refer to a simple X by Y cross, or theprocess of backcrossing, depending on the context.

Gene Conversions

When the term “lettuce plant” is used in the context of the presentinvention, this also includes any gene conversions of that variety. Theterm “gene converted plant” as used herein refers to those lettuceplants which are developed by backcrossing, genetic engineering, ormutation, wherein essentially all of the desired morphological andphysiological characteristics of a variety are recovered in addition tothe one or more genes transferred into the variety via the backcrossingtechnique, genetic engineering, or mutation. Backcrossing methods can beused with the present invention to improve or introduce a characteristicinto the variety. The term “backcrossing” as used herein refers to therepeated crossing of a hybrid progeny back to the recurrent parent,i.e., backcrossing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more times to therecurrent parent. The parental lettuce plant which contributes the genefor the desired characteristic is termed the “nonrecurrent” or “donorparent.” This terminology refers to the fact that the nonrecurrentparent is used one time in the backcross protocol and therefore does notrecur. The parental lettuce plant to which the gene or genes from thenonrecurrent parent are transferred is known as the recurrent parent asit is used for several rounds in the backcrossing protocol. Poehlman &Sleper (1994) and Fehr (1993). In a typical backcross protocol, theoriginal variety of interest (recurrent parent) is crossed to a secondvariety (nonrecurrent parent) that carries the gene of interest to betransferred. The resulting progeny from this cross are then crossedagain to the recurrent parent and the process is repeated until alettuce plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the transferredgene from the nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a trait or characteristic in the original line.To accomplish this, a gene of the recurrent cultivar is modified orsubstituted with the desired gene from the nonrecurrent parent, whileretaining essentially all of the rest of the desired genetic, andtherefore the desired physiological and morphological, constitution ofthe original line. The choice of the particular nonrecurrent parent willdepend on the purpose of the backcross. One of the major purposes is toadd some commercially desirable, agronomically important trait to theplant. The exact backcrossing protocol will depend on the characteristicor trait being altered to determine an appropriate testing protocol.Although backcrossing methods are simplified when the characteristicbeing transferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

Many gene traits have been identified that are not regularly selected inthe development of a new line but that can be improved by backcrossingtechniques. Gene traits may or may not be transgenic. Examples of thesetraits include, but are not limited to, male sterility, modified fattyacid metabolism, modified carbohydrate metabolism, herbicide resistance,resistance for bacterial, fungal, or viral disease, insect resistance,enhanced nutritional quality, industrial usage, yield stability, andyield enhancement. These genes are generally inherited through thenucleus. Several of these gene traits are described in U.S. Pat. Nos.5,777,196, 5,948,957, and 5,969,212, the disclosures of which arespecifically hereby incorporated by reference.

Tissue Culture

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of lettuce andregeneration of plants therefrom is well known and widely published. Forexample, reference may be had to Teng, et al., HortScience, 27:9,1030-1032 (1992); Teng, et al., HortScience, 28:6, 669-1671 (1993);Zhang, et al., Journal of Genetics and Breeding, 46:3, 287-290 (1992);Webb, et al., Plant Cell Tissue and Organ Culture, 38:1, 77-79 (1994);Curtis, et al., Journal of Experimental Botany, 45:279, 1441-1449(1994); Nagata, et al., Journal for the American Society forHorticultural Science, 125:6, 669-672 (2000); and Ibrahim, et al., PlantCell Tissue and Organ Culture, 28(2), 139-145 (1992). It is clear fromthe literature that the state of the art is such that these methods ofobtaining plants are routinely used and have a very high rate ofsuccess. Thus, another aspect of this invention is to provide cellswhich upon growth and differentiation produce lettuce plants having thephysiological and morphological characteristics of variety Cayucos.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, meristematic cells, andplant cells that can generate tissue culture that are intact in plantsor parts of plants, such as leaves, pollen, embryos, roots, root tips,anthers, pistils, flowers, seeds, petioles, suckers, and the like. Meansfor preparing and maintaining plant tissue culture are well known in theart. By way of example, a tissue culture comprising organs has been usedto produce regenerated plants. U.S. Pat. Nos. 5,959,185, 5,973,234, and5,977,445 describe certain techniques, the disclosures of which areincorporated herein by reference.

Additional Breeding Methods

This invention also is directed to methods for producing a lettuce plantby crossing a first parent lettuce plant with a second parent lettuceplant wherein the first or second parent lettuce plant is a lettuceplant of cultivar Cayucos. Further, both first and second parent lettuceplants can come from lettuce cultivar Cayucos. Thus, any such methodsusing lettuce cultivar Cayucos are part of this invention: selfing,backcrosses, hybrid production, crosses to populations, and the like.All plants produced using lettuce cultivar Cayucos as at least oneparent are within the scope of this invention, including those developedfrom cultivars derived from lettuce cultivar Cayucos. Advantageously,this lettuce cultivar could be used in crosses with other, different,lettuce plants to produce the first generation (F₁) lettuce hybrid seedsand plants with superior characteristics. The cultivar of the inventioncan also be used for transformation where exogenous genes are introducedand expressed by the cultivar of the invention. Genetic variants createdeither through traditional breeding methods using lettuce cultivarCayucos or through transformation of cultivar Cayucos by any of a numberof protocols known to those of skill in the art are intended to bewithin the scope of this invention.

The following describes breeding methods that may be used with lettucecultivar Cayucos in the development of further lettuce plants. One suchembodiment is a method for developing cultivar Cayucos progeny lettuceplants in a lettuce plant breeding program comprising: obtaining thelettuce plant, or a part thereof, of cultivar Cayucos, utilizing saidplant or plant part as a source of breeding material, and selecting alettuce cultivar Cayucos progeny plant with molecular markers in commonwith cultivar Cayucos and/or with morphological and/or physiologicalcharacteristics selected from the characteristics listed in Table 1.Breeding steps that may be used in the lettuce plant breeding programinclude pedigree breeding, backcrossing, mutation breeding, andrecurrent selection. In conjunction with these steps, techniques such asRFLP-enhanced selection, genetic marker enhanced selection (for example,SSR markers), and the making of double haploids may be utilized.

Another method involves producing a population of lettuce cultivarCayucos progeny lettuce plants, comprising crossing cultivar Cayucoswith another lettuce plant, thereby producing a population of lettuceplants, which, on average, derive 50% of their alleles from lettucecultivar Cayucos. A plant of this population may be selected andrepeatedly selfed or sibbed with a lettuce cultivar resulting from thesesuccessive filial generations. One embodiment of this invention is thelettuce cultivar produced by this method and that has obtained at least50% of its alleles from lettuce cultivar Cayucos.

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see Fehr and Walt, Principles of CultivarDevelopment, pp. 261-286 (1987). Thus the invention includes lettucecultivar Cayucos progeny lettuce plants comprising a combination of atleast two cultivar Cayucos traits selected from the group consisting ofthose listed in Table 1 or the cultivar Cayucos combination of traitslisted in the Summary of the Invention, so that said progeny lettuceplant is not significantly different for said traits than lettucecultivar Cayucos as determined at the 5% significance level when grownin the same environmental conditions. Using techniques described herein,molecular markers may be used to identify said progeny plant as alettuce cultivar Cayucos progeny plant. Mean trait values may be used todetermine whether trait differences are significant, and preferably thetraits are measured on plants grown under the same environmentalconditions. Once such a variety is developed, its value is substantialsince it is important to advance the germplasm base as a whole in orderto maintain or improve traits such as yield, disease resistance, pestresistance, and plant performance in extreme environmental conditions.

Progeny of lettuce cultivar Cayucos may also be characterized throughtheir filial relationship with lettuce cultivar Cayucos, as for example,being within a certain number of breeding crosses of lettuce cultivarCayucos. A breeding cross is a cross made to introduce new genetics intothe progeny, and is distinguished from a cross, such as a self or a sibcross, made to select among existing genetic alleles. The lower thenumber of breeding crosses in the pedigree, the closer the relationshipbetween lettuce cultivar Cayucos and its progeny. For example, progenyproduced by the methods described herein may be within 1, 2, 3, 4, or 5breeding crosses of lettuce cultivar Cayucos.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cell tissue cultures from which lettuce plants can beregenerated, plant calli, plant clumps, and plant cells that are intactin plants or parts of plants, such as leaves, pollen, embryos,cotyledons, hypocotyl, roots, root tips, anthers, pistils, flowers,ovules, seeds, stems, and the like.

TABLE

Table 2 below compares some of the characteristics of leaf lettucecultivar Cayucos with the commercial lettuce cultivar, Dragoon. Column 1lists the characteristics, column 2 shows the characteristics forlettuce cultivar Cayucos, and column 3 shows the characteristics forlettuce cultivar Dragoon.

TABLE 2 Characteristic Cayucos Dragoon Plant size Small Very small Topclosing Medium closed Closed Leaf color Medium green Medium to darkgreen

DEPOSIT INFORMATION

A deposit of the lettuce cultivar Cayucos disclosed above and recited inthe appended claims has been made with the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va. 20110. Thedate of deposit was May 4, 2011. The deposit of 2,500 seeds was takenfrom the same deposit maintained by Enza Zaden USA Inc. since prior tothe filing date of this application. All restrictions will beirrevocably removed upon granting of a patent, and the deposit isintended to meet all of the requirements of 37 C.F.R. §§1.801-1.809. TheATCC Accession Number is PTA-11867. The deposit will be maintained inthe depository for a period of thirty years, or five years after thelast request, or for the enforceable life of the patent, whichever islonger, and will be replaced as necessary during that period.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions, and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions, and sub-combinations as are within their truespirit and scope.

What is claimed is:
 1. A seed of lettuce cultivar Cayucos, wherein arepresentative sample seed of said cultivar was deposited under ATCCAccession No. PTA-11867.
 2. A lettuce plant, or a part thereof, producedby growing the seed of claim
 1. 3. A tissue culture produced fromprotoplasts or cells from the plant of claim 2, wherein said cells orprotoplasts are produced from a plant part selected from the groupconsisting of leaf, pollen, embryo, cotyledon, hypocotyl, meristematiccell root, root tip, pistil, anther, ovule, flower, shoot, stem, seed,and petiole.
 4. A lettuce plant regenerated from the tissue culture ofclaim 3, wherein the plant has all of the morphological andphysiological characteristics of cultivar Cayucos.
 5. A method forproducing a lettuce seed comprising crossing two lettuce plants andharvesting the resultant lettuce seed, wherein at least one lettuceplant is the lettuce plant of claim
 2. 6. A lettuce seed produced by themethod of claim
 5. 7. A lettuce plant, or a part thereof, produced bygrowing said seed of claim
 6. 8. The method of claim 5, wherein at leastone of said lettuce plants is transgenic.
 9. A method of producing amale sterile lettuce plant, wherein the method comprises introducing anucleic acid molecule that confers male sterility into the lettuce plantof claim
 2. 10. A male sterile lettuce plant produced by the method ofclaim
 9. 11. A method of producing an herbicide resistant lettuce plant,wherein said method comprises introducing a gene conferring herbicideresistance into the plant of claim 2, wherein the gene is selected fromthe group consisting of glyphosate, sulfonylurea, imidazolinone,dicamba, glufosinate, phenoxy proprionic acid, L-phosphinothricin,cyclohexone, cyclohexanedione, triazine, and benzonitrile.
 12. Anherbicide resistant lettuce plant produced by the method of claim 11.13. A method of producing a pest or insect resistant lettuce plant,wherein said method comprises introducing a gene conferring pest orinsect resistance into the plant of claim
 2. 14. A pest or insectresistant lettuce plant produced by the method of claim
 13. 15. Thelettuc plant of claim 14, wherein the gene encodes a Bacillusthuringiensis endotoxin.
 16. A method of producing a disease resistantlettuce plant, wherein said method comprises introducing a geneconferring disease resistance into the plant of claim
 2. 17. A diseaseresistant lettuce plant produced by the method of claim
 16. 18. A methodof producing a lettuce plant with a value-added trait, wherein saidmethod comprises introducing a gene conferring a value-added trait intothe plant of claim 2, where said gene encodes a protein selected fromthe group consisting of a ferritin, a nitrate reductase, and a monellin.19. A lettuce plant with a value-added trait produced by the method ofclaim
 18. 20. A method of introducing a desired trait into lettucecultivar Cayucos wherein the method comprises: (a) crossing a Cayucosplant, wherein a representative sample of seed was deposited under ATCCAccession No. PTA-11867, with a plant of another lettuce cultivar thatcomprises a desired trait to produce progeny plants wherein the desiredtrait is selected from the group consisting of male sterility, herbicideresistance, insect or pest resistance, modified bolting and resistanceto bacterial disease, fungal disease or viral disease; (b) selecting oneor more progeny plants that have the desired trait to produce selectedprogeny plants; (c) crossing the selected progeny plants with theCayucos plant to produce backcross progeny plants; (d) selecting forbackcross progeny plants that have the desired trait and all of thephysiological and morphological characteristics of lettuce cultivarCayucos listed in Table 1; and (e) repeating steps (c) and (d) two ormore times in succession to produce selected third or higher backcrossprogeny plants that comprise the desired trait and all of thephysiological and morphological characteristics of lettuce cultivarCayucos listed in Table
 1. 21. A lettuce plant produced by the method ofclaim 20, wherein the plant has the desired trait.
 22. The lettuce plantof claim 21, wherein the desired trait is herbicide resistance and theresistance is conferred to an herbicide selected from the groupconsisting of glyphosate, sulfonylurea, imidazolinone, dicamba,glufosinate, phenoxy proprionic acid, L-phosphinothricin, cyclohexone,cyclohexanedione, triazine, and benzonitrile.
 23. The lettuce plant ofclaim 21, wherein the desired trait is insect or pest resistance and theinsect or pest resistance is conferred by a transgene encoding aBacillus thuringiensis endotoxin.